All
primers used in high throughput are labelled with 6-nucleotide barcode sequences in the
5’ end of the primer sequences listed in the table below.
For
the variable region of IgG and IgA heavy chains, two degenerate primers for mice (mH1
and mH2) are used together as one forward primer, the reverse primers on
constant region ensure the specificity of IgG or IgA heavy chain class.
Primer name |
Sequence |
mVK-Forward |
GACATTCAGCTGACCCAGTCTCCA |
mH1-Forward |
SARGTNMAGCTGSAGSAGTC |
mH2-Forward |
SARGTNMAGCTGSAGSAGTCWGG |
mVK-Reverse |
AGNTTGGTTCCACCRCCGAACG |
mVG-Reverse |
GATAGACHGATGGGGSTGTYGTT |
mVA-reverse |
TGGTGGGATTTCTCGCAGAC |
No. | FW Primer | FW Barcode | Nucleotides | RW Primer | RW Barcode | Nucleotides | Sample Name |
1 | mVK-BC01F | barcode1 | ATCACG | mVK-BC10R | barcode10 | TAGCTT | μMT-Spleen-PBS-2-Igk |
2 | mVK-BC01F | barcode1 | ATCACG | mVK-BC11R | barcode11 | GGCTAC | μMT-Liver-PBS-1-Igk |
3 | mVK-BC01F | barcode1 | ATCACG | mVK-BC12R | barcode12 | CTTGTA | μMT-Liver-PBS-2-Igk |
4 | mVK-BC01F | barcode1 | ATCACG | mVK-BC14R | barcode14 | AGTTCC | μMT-Liver-PBS-4-Igk |
5 | mVK-BC01F | barcode1 | ATCACG | mVK-BC15R | barcode15 | ATGTCA | μMT-Liver-NP-6-Igk |
6 | mVK-BC01F | barcode1 | ATCACG | mVK-BC16R | barcode16 | CCGTCC | μMT-Liver-NP-7-Igk |
7 | mVK-BC01F | barcode1 | ATCACG | mVK-BC18R | barcode18 | GTCCGC | μMT-Liver-NP-9-Igk |
8 | mVK-BC01F | barcode1 | ATCACG | mVK-BC20R | barcode20 | GTGGCC | μMT-Lung-PBS-1-Igk |
9 | mVK-BC01F | barcode1 | ATCACG | mVK-BC21R | barcode21 | GTTTCG | μMT-Lung-PBS-2-Igk |
10 | mVK-BC01F | barcode1 | ATCACG | mVK-BC22R | barcode22 | CGTACG | μMT-Lung-PBS-3-Igk |
11 | mVK-BC01F | barcode1 | ATCACG | mVK-BC23R | barcode23 | GAGTGG | μMT-Lung-PBS-4-Igk |
12 | mVK-BC01F | barcode1 | ATCACG | mVK-BC24R | barcode24 | GGTAGC | μMT-Lung-NP-6-Igk |
13 | mVK-BC01F | barcode1 | ATCACG | mVK-BC27R | barcode27 | ATTCCT | μMT-Lung-NP-10-Igk |
14 | mVK-BC01F | barcode1 | ATCACG | mVK-BC28R | barcode28 | CAAAAG | μMT-Kidney-PBS-1-Igk |
15 | mVK-BC01F | barcode1 | ATCACG | mVK-BC29R | barcode29 | CAACTA | μMT-Kidney-PBS-2-Igk |
16 | mVK-BC01F | barcode1 | ATCACG | mVK-BC32R | barcode32 | CACTCA | μMT-Kidney-NP-6-Igk |
17 | mVK-BC01F | barcode1 | ATCACG | mVK-BC34R | barcode34 | CATGGC | μMT-Kidney-NP-9-Igk |
18 | mVK-BC01F | barcode1 | ATCACG | mVK-BC37R | barcode37 | CGGAAT | BALBc-Kidney-PBS-5-Igk |
19 | mVK-BC01F | barcode1 | ATCACG | mVK-BC38R | barcode38 | CTAGCT | BALBc-Kidney-NP-6-Igk |
20 | mVK-BC01F | barcode1 | ATCACG | mVK-BC39R | barcode39 | CTATAC | BALBc-Kidney-NP-7-Igk |
21 | mVK-BC01F | barcode1 | ATCACG | mVK-BC40R | barcode40 | CTCAGA | BALBc-Kidney-NP-9-Igk |
22 | mVK-BC01F | barcode1 | ATCACG | mVK-BC41R | barcode41 | GACGAC | BALBc-Lung-PBS-4-Igk |
23 | mVK-BC01F | barcode1 | ATCACG | mVK-BC42R | barcode42 | TAATCG | BALBc-Lung-PBS-5-Igk |
24 | mVK-BC01F | barcode1 | ATCACG | mVK-BC43R | barcode43 | TACAGC | BALBc-Lung-NP-6-Igk |
25 | mVK-BC01F | barcode1 | ATCACG | mVK-BC44R | barcode44 | TATAAT | BALBc-Lung-NP-8-Igk |
26 | mVK-BC01F | barcode1 | ATCACG | mVK-BC45R | barcode45 | TCATTC | μMT-Lung-NP-6-Igk |
27 | mVK-BC01F | barcode1 | ATCACG | mVK-BC46R | barcode46 | TCCCGA | μMT-Lung-NP-12-Igk |
28 | mVK-BC01F | barcode1 | ATCACG | mVK-BC47R | barcode47 | TCGAAG | BALBc-Spleen-PBS-Igk |
29 | mVK-BC02F | barcode2 | CGATGT | mVK-BC10R | barcode10 | TAGCTT | μMT_CD3-T_Sort1-Igk |
30 | mVK-BC02F | barcode2 | CGATGT | mVK-BC11R | barcode11 | GGCTAC | μMT_CD3-T_Sort2-Igk |
31 | mVK-BC02F | barcode2 | CGATGT | mVK-BC12R | barcode12 | CTTGTA | μMT_CD3-T_Sort3-Igk |
32 | mVK-BC02F | barcode2 | CGATGT | mVK-BC13R | Barcode13 | AGTCAA | μMT_CD4-T_PBS4-Igk |
33 | mVK-BC02F | barcode2 | CGATGT | mVK-BC14R | barcode14 | AGTTCC | μMT_CD8-T_PBS4-Igk |
34 | mVK-BC02F | barcode2 | CGATGT | mVK-BC15R | barcode15 | ATGTCA | μMT_CD4-T_NP7-Igk |
35 | mVK-BC02F | barcode2 | CGATGT | mVK-BC16R | barcode16 | CCGTCC | μMT_CD8-T_NP7-Igk |
36 | mVK-BC02F | barcode2 | CGATGT | mVK-BC17R | barcode17 | GTAGAG | μMT_CD4-T_NP8-Igk |
37 | mVK-BC02F | barcode2 | CGATGT | mVK-BC18R | barcode18 | GTCCGC | μMT_CD8-T_NP8-Igk |
38 | mVK-BC02F | barcode2 | CGATGT | mVK-BC19R | barcode19 | GTGAAA | μMT_CD4-T_NP12-Igk |
39 | mVK-BC02F | barcode2 | CGATGT | mVK-BC20R | barcode20 | GTGGCC | μMT_CD8-T_NP12-Igk |
40 | mVK-BC02F | barcode2 | CGATGT | mVK-BC21R | barcode21 | GTTTCG | BALBc_CD4-T_PBS-Igk |
41 | mVK-BC02F | barcode2 | CGATGT | mVK-BC22R | barcode22 | CGTACG | BALBc_CD8-T_PBS-Igk |
42 | mVK-BC02F | barcode2 | CGATGT | mVK-BC23R | barcode23 | GAGTGG | BALBc_B_PBS-Igk |
43 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC11R | barcode11 | GGCTAC | μMT_Liver_PBS2_Igγ |
44 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC12R | barcode12 | CTTGTA | μMT_Liver_PBS3_Igγ |
45 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC13R | Barcode13 | AGTCAA | μMT_Liver_PBS4_Igγ |
46 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC14R | barcode14 | AGTTCC | μMT_Liver_NP6_Igγ |
47 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC15R | barcode15 | ATGTCA | μMT_Liver_NP7_Igγ |
48 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC16R | barcode16 | CCGTCC | μMT_Liver_NP8_Igγ |
49 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC17R | barcode17 | GTAGAG | μMT_Liver_NP9_Igγ |
50 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC18R | barcode18 | GTCCGC | μMT_Lung_PBS1_Igγ |
51 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC21R | barcode21 | GTTTCG | μMT_Lung_PBS4_Igγ |
52 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC23R | barcode23 | GAGTGG | μMT_Lung_NP7_Igγ |
53 | mH1/2-BC04F | barcode4 | TGACCA | mVG-BC26R | barcode26 | ATGAGC | μMT_Kidney_PBS1_Igγ |
54 | mH1/2-BC05F | barcode5 | ACAGTG | mVG-BC10R | barcode10 | TAGCTT | μMT_Kidney_NP6_Igγ |
55 | mH1/2-BC05F | barcode5 | ACAGTG | mVG-BC11R | barcode11 | GGCTAC | μMT_Kidney_NP7_Igγ |
56 | mH1/2-BC05F | barcode5 | ACAGTG | mVG-BC17R | barcode17 | GTAGAG | μMT_Spleen_PBS2_Igγ |
57 | mH1/2-BC05F | barcode5 | ACAGTG | mVG-BC18R | barcode18 | GTCCGC | BALBc_Spleen_PBS_Igγ |
58 | mH1/2-BC05F | barcode5 | ACAGTG | mVG-BC21R | barcode21 | GTTTCG | BALBc_Kidney_PBS4-Sorted_Igγ |
59 | mH1/2-BC05F | barcode5 | ACAGTG | mVG-BC23R | barcode23 | GAGTGG | BALBc_Kidney_NP7-Sorted_Igγ |
60 | mH1/2-BC05F | barcode5 | ACAGTG | mVG-BC24R | barcode24 | GGTAGC | BALBc_Kidney_NP8-Sorted_Igγ |
61 | mH1/2-BC05F | barcode5 | ACAGTG | mVG-BC27R | barcode27 | ATTCCT | BALBc_Lung_PBS1-Sorted_Igγ |
62 | mH1/2-BC05F | barcode5 | ACAGTG | mVG-BC28R | barcode28 | CAAAAG | BALBc_Lung_PBS3-Sorted_Igγ |
63 | mH1/2-BC05F | barcode5 | ACAGTG | mVG-BC29R | barcode29 | CAACTA | BALBc_Lung_NP6-Sorted_Igγ |
64 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC11R | barcode11 | GGCTAC | Lung_NP10-Sorted_Igγ |
65 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC12R | barcode12 | CTTGTA | BALBc_CD4+T_PBS-Sorted_Igγ |
66 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC13R | Barcode13 | AGTCAA | BALBc_CD8+T_PBS-Sorted_Igγ |
67 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC14R | barcode14 | AGTTCC | BALBc_B_PBS-Sorted_Igγ |
68 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC15R | barcode15 | ATGTCA | BALBc_CD4+T_NP7-Sorted_Igγ |
69 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC17R | barcode17 | GTAGAG | BALBc_Macrophage_PBS1-Sorted_Igγ |
70 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC18R | barcode18 | GTCCGC | BALBc_Macrophage_PBS2-Sorted_Igγ |
71 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC19R | barcode19 | GTGAAA | BALBc_Macrophage_PBS3-Sorted_Igγ |
72 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC20R | barcode20 | GTGGCC | μMT_CD4+T_PBS4_Igγ |
73 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC21R | barcode21 | GTTTCG | μMT_CD8+T_PBS4_Igγ |
74 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC22R | barcode22 | CGTACG | μMT_CD4+T_NP8_Igγ |
75 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC23R | barcode23 | GAGTGG | μMT_CD8+T_NP8_Igγ |
76 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC24R | barcode24 | GGTAGC | μMT_CD4+T_NP12_Igγ |
77 | mH1/2-BC06F | barcode6 | GCCAAT | mVG-BC25R | barcode25 | ACTGAT | μMT_CD8+T_NP12_Igγ |
78 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC30R | barcode30 | CACCGG | μMT_Liver_PBS1_Igα |
79 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC31R | barcode31 | CACGAT | μMT_Liver_PBS2_Igα |
80 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC32R | barcode32 | CACTCA | μMT_Liver_PBS3_Igα |
81 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC33R | barcode33 | CAGGCG | μMT_Liver_PBS4_Igα |
82 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC34R | barcode34 | CATGGC | μMT_Liver_NP6_Igα |
83 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC35R | barcode35 | CATTTT | μMT_Liver_NP7_Igα |
84 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC37R | barcode37 | CGGAAT | μMT_Liver_NP9_Igα |
85 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC38R | barcode38 | CTAGCT | μMT_Lung_PBS1_Igα |
86 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC39R | barcode39 | CTATAC | μMT_Lung_PBS2_Igα |
87 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC40R | barcode40 | CTCAGA | μMT_Lung_PBS3_Igα |
88 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC41R | barcode41 | GACGAC | μMT_Lung_PBS4_Igα |
89 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC42R | barcode42 | TAATCG | μMT_Lung_NP6_Igα |
90 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC46R | barcode46 | TCCCGA | μMT_Kidney_PBS1_Igα |
91 | mH1/2-BC04F | barcode4 | TGACCA | mVA-BC47R | barcode47 | TCGAAG | μMT_Kidney_PBS2_Igα |
92 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC33R | barcode33 | CAGGCG | μMT_Kidney_NP8_Igα |
93 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC34R | barcode34 | CATGGC | μMT_Kidney_NP9_Igα |
94 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC35R | barcode35 | CATTTT | μMT_Liver_NP10_Igα |
95 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC37R | barcode37 | CGGAAT | μMT_Kidney_NP10_Igα |
96 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC39R | barcode39 | CTATAC | BALBc_Spleen_PBS_Igα |
97 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC40R | barcode40 | CTCAGA | BALBc_Kidney_PBS2-Sorted_Igα |
98 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC41R | barcode41 | GACGAC | BALBc_Kidney_PBS3-Sorted_Igα |
99 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC42R | barcode42 | TAATCG | BALBc_Kidney_PBS4-Sorted_Igα |
100 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC43R | barcode43 | TACAGC | BALBc_Kidney_PBS5-Sorted_Igα |
101 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC44R | barcode44 | TATAAT | BALBc_Kidney_NP7-Sorted_Igα |
102 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC45R | barcode45 | TCATTC | BALBc_Kidney_NP7-Sorted_Igα |
103 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC46R | barcode46 | TCCCGA | BALBc_Kidney_NP9-Sorted_Igα |
104 | mH1/2-BC05F | barcode5 | ACAGTG | mVA-BC48R | barcode48 | TCGGCA | μMT_Lung_PBS1-Sorted_Igα |
105 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC30R | barcode30 | CACCGG | μMT_Lung_PBS3-Sorted_Igα |
106 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC31R | barcode31 | CACGAT | μMT_Lung_NP6-Sorted_Igα |
107 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC33R | barcode33 | CAGGCG | μMT_Lung_NP10-Sorted_Igα |
108 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC34R | barcode34 | CATGGC | BALBc_CD4+T_PBS_Igα |
109 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC35R | barcode35 | CATTTT | BALBc_CD8+T_PBS_Igα |
110 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC36R | barcode36 | CCAACA | BALBc_B_PBS_Igα |
111 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC37R | barcode37 | CGGAAT | BALBc_CD4+T_NP7_Igα |
112 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC38R | barcode38 | CTAGCT | BALBc_CD8+T_NP7_Igα |
113 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC39R | barcode39 | CTATAC | BALBc_Macrophage_PBS1_Igα |
114 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC40R | barcode40 | CTCAGA | BALBc_Macrophage_PBS2_Igα |
115 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC41R | barcode41 | GACGAC | BALBc_Macrophage_PBS3_Igα |
116 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC43R | barcode43 | TACAGC | μMT_CD8+T_PBS4_Igα |
117 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC44R | barcode44 | TATAAT | μMT_CD4+T_NP8_Igα |
118 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC45R | barcode45 | TCATTC | μMT_CD8+T_NP8_Igα |
119 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC46R | barcode46 | TCCCGA | μMT_CD4+T_NP12_Igα |
120 | mH1/2-BC06F | barcode6 | GCCAAT | mVA-BC47R | barcode47 | TCGAAG | μMT_CD8+T_NP12_Igα |
Homo Sapiens (Human) Primers (used in Sanger sequencing)
Ig heavy chain
Nest PCR or half-nest PCR
First round:
VH1 (5'-GAGGTGCAGCTCGAGGAGTCTGGG-3'),
VH2 (5'-CAGGTGCAGCTCGAGCAGTCTGGG-3'),
VH3 (5'-CAGGTACAGCTCGAGCAGTCAGG-3'),
VH4 (5'-CAGGTGCAGCTGCTCGAGTCGGG-3'),
Cμ-CH1, 5'-ACGCTGC TCGTATCCGACGGG-3'
Or CγCH1, 5'-ACGCTGCTCGTATCCGACGGG-3'
Second round:
FR2 coupled with JH primer was used
FR2: 5’-TGG(A/G)TCCG(A/C/G)CAG(G/C)C(T/C)CC(A/C/G/T)GG-3’;
TGGRTCCGVCAGSCYCCNGG
JH: 5’-AACTGCAGAGGAGACGGTGACC-3’
or JH2: 5’-GTGACCAGGGTNCCTTGGCCCCAAG-3'
Ig kappa chain
Vκ1 (5’-GACATCGAGCTCACCCAGTCTCC-3’) and
Vκ 2 (5’-GAAATTGAGCTCACGCAGTCTCCA-3’)
Cκ2 primer (Cκ2: 5’-CGGGAAGATGAAGACAGATGGTGC-3’)
Half-nest PCR
Cκ1 primer (5’-TGGTGCAGCCACAGTTCGTTT-3’)
Or
JK1/2/3/4 : 5’-CTTACGTTTGATCTCCACCTTGGTCCC-3’
JK5 : 5’-CTTACGTTTAATCTCCAGTCGTGTCCC-3’